1. Field of the Invention
The present invention relates generally to total internal reflection fluorescence microscopy (TIRFM) and, more particularly, to total internal reflection fluorescence microscopes which use conventional white-light sources which can also be used as a conventional microscope.
2. Prior Art
Total internal reflection is an optical phenomenon. When light strikes the interface between two optical media of different refractive indices, the light incident at an angle greater than the xe2x80x9ccritical anglexe2x80x9d undergoes xe2x80x9ctotal reflectionxe2x80x9d. However, beyond the angle of total reflection, the electromagnetic field of the incoming/reflected light extends into the Z-direction as can be seen from FIG. 1. FIG. 1 illustrates a laser 100 incident on an interface between two materials 102, 104 having refractive indices n1 and n2 respectively. The laser 100 is reflected at the interface while a wave 106 extends into the z-direction. The strength of the field in the z-direction is termed an xe2x80x9cevanescent wavexe2x80x9d 106 which decreases exponentially and its effects extend only a few hundred nano-meters into the second medium 104.
That portion of a specimen within the evanescent field can be excited to emit fluorescence and consequently can be seen or recorded. This is the essence of TIRFM. TIRFM is an optical technique used to observe single molecule fluorescence which has been used for many years by biophysicists. TIRFM is also gaining popularity with cell biologist and nueroscientists to observe membrane fluorescence, in part because of the development of membrane specific dyes.
The condition for total reflection is:
xcex8xe2x89xa7sinxe2x88x921(n2/n1)xe2x80x83xe2x80x83(1)
where xcex8 is the angle of incidence of the light, n1, is the refractive index of the first optical medium, n2 is the refractive index of the second optical medium, and n2xe2x89xa7n1.
The condition of equation 1 is identical to that of creating an evanescent wave. The key advantage of TIRFM is the shallow penetration depth of the evanescent wave. Only fluorophore molecules very near the surface of a specimen are excited to emit, creating a super thin optical section. Outside of the evanescent field, fluorescence is minimal which leads to images of very high contrast.
To create the condition to total reflection of equation 1, a TIRFM microscope having a laser light source directs laser light 200 at a specimen at the angle xcex8 needed for total reflection as shown in FIG. 2. Conventional white-light sources used in conventional microscopes (other than TIRFM microscopes) such as mercury, halogen, xenon, and metal halide lamps have not been used in TIRFM microscopes because they do not illuminate the angle xcex8 or higher needed for totally reflected illumination. Therefore, expensive microscopes are needed having laser light sources which are dedicated to TIRFM. Furthermore, the laser light sources generally have a limited wavelength and narrow bandwidth.
Therefore it is an object of the present invention to provide a microscope capable of TIRFM with conventional white-light sources.
It is a further object of the present invention to provide a microscope capable of both TIRFM and conventional microscopy with conventional white-light sources.
It is yet a further object of the present invention to provide a microscope capable of TIRFM having a conventional white-light source which is inexpensive as compared to conventional TIRFM microscopes employing a laser light source.
It is still yet a further object of the present invention to provide a microscope capable of TIRFM which can operate over a wider wavelength bandwidth than conventional TIRFM microscopes employing laser light sources.
Accordingly, a microscope is provided. The microscope comprises: a first optical path having; a white-light source, an illumination optical system, and an excitation filter disposed in said illumination optical system; and an observation optical path having; an objective lens, an emission filter, a dichromatic mirror disposed on a cross point of said first optical path and said observation optical path; and an annular slit member disposed in said illumination optical system.
Also provided is a microscope for use in total internal reflection fluorescence microscopy (TIRFM). The microscope comprises: a first white-light source for directing light along a first optical path; an annular slit member disposed in the first optical path, the annular slit member having an annular slit for blocking all but an annulus of light corresponding to the annular slit; and an objective lens for directing the annulus of light to a specimen such that TIRFM of the specimen is achieved.
Preferably, the microscope further comprises converting means for converting the microscope to and from a conventional microscope. The converting means preferably comprises one of:
(1) a mechanism upon which the annular slit member is disposed, the mechanism for moving the annular slit member into and out from the first optical path;
(2) a second white-light source for directing light along a second optical path; a blocking means disposed in each of the first and second optical paths for selectively blocking light from either the first or second optical paths; and a beam splitter disposed at the convergence of the first and second optical paths to transmit one of the annulus of light from the first optical path or the light from the second optical path to the objective lens; or
(3) a turret having at least two stations, each station being capable of being selectively disposed in the first optical path and having a means to direct light from the first optical path to the objective lens, one of the stations further having a diffuser for diffusing the annulus of light.
Further provided is a combined total internal reflection fluorescence microscopy (TIRFM) and conventional microscope. The combined microscope comprises: a first white-light source for directing light along a first optical path; an annular slit member disposed in the first optical path, the annular slit member having an annular slit for blocking all but an annulus of light corresponding to the annular slit; an objective lens for directing the annulus of light to a specimen such that TIRFM of the specimen is achieved; and converting means for converting the microscope to and from a TIRFM microscope and a conventional microscope.
The microscope/combined microscope are preferably configured with either an upright set-up configuration or an inverted set-up configuration.
Still further provided is an annular slit member for use in a microscope to permit total internal reflection fluorescence microscopy (TIRFM). The annular slit member has an annular slit for blocking all but an annulus of light corresponding to the annular slit. The annular slit member preferably further comprises a mechanism for housing the annular slit member for selective movement thereof between a first position in the optical path of a white-light source and a second position removed from the first optical path.
Still yet further provided is a method for converting a conventional microscope into a total internal reflection fluorescence microscopy (TIRFM) microscope. The method comprises the steps of: providing a conventional microscope; and disposing an annular slit member in an optical path of the microscope between a white-light source and an objective lens, the annular slit member having an annular slit for blocking all but an annulus of light corresponding to the annular slit.
The disposing step preferably comprises either:
(1) fixing the annular slit member to the microscope in the optical path; or
(2) fixing the annular slit member to a mechanism which is movably fixed to the microscope for selective movement between a first position where the annular slit member is in the first optical path and a second position where the annular slit member is removed from the first optical path.